PET54 and PET122 are nuclear genes that control expression of mitochondrial cytochrome c oxidase genes in S. cerevisiae. The two general aims of this proposal are I) to understand the biochemical function of the PET54 gene product and II) to study the regulation of the PET122 gene, and in particular, the role of an antisense transcript that is produced in vivo from the PET122 locus. The protein encoded by PET54 is particularly interesting because it performs two seemingly different functions in mitochondrial gene expression. It promotes translation of COX3 mRNA and splicing of COX1 pre- mRNA. The COX3 mRNA translation function is performed in cooperation with the PET494 and PET122 gene products. In COX1 gene expression, the PET54 protein promotes excision of intron aI5beta from COX1 pre-mRNA. In this case, the PET54 protein cooperates with the MSS18 and MRS1 gene products. Genetic evidence indicates that the PET54 protein is bifunctional. The FIRST SPECIFIC AIM is to genetically dissect the functional domains required for expression of COX1 and COX3, respectively. The SECOND SPECIFIC AIM is to assess whether Pet54p is part of a multisubunit protein, and to examine whether copurified polypeptides are products of the PET494, PET122, MSS18, or MRS1 genes. Analytical scale purification of the native form of Pet54p will be accomplished by immunoaffinity chromatography. A monoclonal antibody to an epitope "tag", which was added to a non-essential region of the Pet54 polypeptide, will be used. The THIRD SPECIFIC AIM is to attempt protein-dependent in vitro splicing of COX1 pre-mRNA bearing intron aI5beta and the FOURTH SPECIFIC AIM is to test whether the native Pet54 protein possesses RNA binding activity. Modulation of COX3 gene expression, apparently at the translational level, has been observed when yeast are released from glucose repression. A possible mechanism for this regulation of COX3 gene expression is to regulate the expression of PET122, PET54 and/or PET494, which are known to promote the translational of COX3 mRNA. A particularly interesting facet of PET122 gene expression is the presence of a natural antisense transcript in vivo. The FIFTH SPECIFIC AIM is to carefully study the transcriptional regulation of both the PET122 mRNA and the antisense RNA. The SIXTH SPECIFIC AIM is to assess whether PET122 expression is modulated at a post- transcriptional level by the "antisense" transcript. Taken together, the studies proposed here will elucidate several mechanisms for post- transcriptional control of gene expression in eukaryotes. Furthermore, they will improve our understanding of mechanisms of nuclear-mitochondrial genetic interactions, which are essential for biogenesis of respiratory- competent mitochondria, which in turn are essential for viability of most eukaryotic organisms.